Osteocytes, the cells that reside within bone matrix, are the most abundant bone cells. They function as the mechanical sensors in bone, and are critical to activation and coordination of osteoclastic and osteoblastic activities by which bone adapts to mechanical usage, maintains its health and prevents fractures. The mechanisms underlying osteocyte mechanotransduction are not well understood, though changes osteocyte mechanosensitivity have been implicated in regulating the effect of both bone anabolic agents and sex hormones. We have developed engineering models which show that small whole bone strains can be amplified locally around osteocyte processes by focal attachments to the canalicular wall. Osteocyte cell bodies cannot see similar high strains as they are too compliant and lack the cellular attachments needed for local strain amplification. These mathematical models argue that the osteocyte cell process may be uniquely designed to function as a detector of small tissue strains. To test this hypothesis, we developed a broad-based multiple-PI program that combines expertise in ion channel physiology, in vivo osteocyte structure/biomechanics and bioengineering/modeling to understand how osteocytes perceive and transduce their local mechanical environment. This program will a) examine the functional polarity of osteocyte mechano- responsiveness using electrophysiological approaches on cultured osteocytes (Aim 1), b) identify the molecular components of mechanotransduction complexes in osteocytes (Aim 2), c) characterize the structure of the mechanotransduction complex in osteocytes in vivo (Aim 3) and d) build integrative mathematical models relating local hydrodynamic forces and membrane strains at osteocyte processes and cell bodies to cellular responses in vitro and in vivo (Aim 4). We have also developed a novel technology (Stokesian Fluid Stimulus probe) that allows us to hydrodynamically load osteocyte processes vs. cell bodies at extremely low forces (<10pN) typical of what bone cells actually experience in vivo. Expansion of this technology to interrogate mechano-responsiveness in a broad range of cell types is a developmental goal of this grant. Significance: Understanding how osteocytes perceive and transduce mechanical signals may provide key new insights into bone physiology leading to the identification of novel therapeutic targets against bone loss due to aging and disease.